PROJECT SUMMARY/ABSTRACT G protein-coupled receptors (GPCRs) are important targets of hormones, neurotransmitters and approximately one-third of FDA-approved drugs. These receptors signal by coupling to transducer proteins from four families of heterotrimeric G proteins and a family of four arrestins. It is now recognized that individual GPCRs produce complex signaling responses by interacting with several different transducer subtypes, i.e. that signaling is ?pluridimensional?. The broad selectivity of a given GPCR for different transducer proteins defines it's ability to influence cellular behavior, therefore it is important to know which tranducers can be activated by each receptor. This information can be surprisingly difficult to obtain, in large part because transducer coupling is most often inferred indirectly using assays that monitor events that are well downstream from receptors, such as accumulation of second messengers. It is also understood that certain activating ligands (agonists) can specifically promote GPCR coupling to some transducers at the expense of others, a phenomenon most often referred to as ?biased agonism?. Biased agonists are especially promising candidates for drug development, and have rapidly advanced into the clinical setting, because they are able to produce therapeutic responses without also producing adverse effects. Almost all biased agonists discovered to date select between G proteins and arrestins as classes, and there are very few ligands that are thought to select between different G protein subtypes. Again, a critical roadblock to discovery and development of G protein subtype-selective drugs is the reliance on downstream signaling assays, which lack the necessary sensitivity to detect subtle ligand bias and are susceptible to signal crosstalk. We will address these problems using a combination of advanced optical tools that allow direct assessment of receptor-transducer coupling. Specifically, we will use improved cellular and cell-free assays based on engineered mini G (mG) proteins to: 1) comprehensively profile GPCR transducer coupling for >200 non-sensory GPCRs, and 2) characterize and discover new G protein subtype-selective ligands. These studies will generate and test hypotheses that are central to pluridimensional signaling, will make a powerful new set of sensitive transducer assays available to the scientific community, and will advance understanding of G protein subtype-biased ligands as potential therapeutic agents.